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Human adenovirus (HAdV) can cause severe disease and death in both immunocompromised and immunocompetent patients. The current standards of treatment are often ineffective, and no approved antiviral therapy against HAdV exists. We report here the design and validation of a fluorescence-based high-content screening platform for the identification of novel anti-HAdV compounds. The screen was conducted using a wildtype-like virus containing the red fluorescent protein (RFP) gene under the regulation of the HAdV major late promoter. Thus, RFP expression allows monitoring of viral late gene expression (a surrogate marker for virus replication), and compounds affecting virus growth can be easily discovered by quantifying RFP intensity. We used our platform to screen ~1200 FDA-approved small molecules, and identified several cardiotonic steroids, corticosteroids and chemotherapeutic agents as anti-HAdV compounds. Our screening platform provides the stringency necessary to detect compounds with varying degrees of antiviral activity, and facilitates drug discovery/repurposing to combat HAdV infections.

We have determined the effect of seven serine- and arginine-rich (SR) proteins and 15 heterogenous nuclear ribonucleoproteins (hnRNPs) on human papillomavirus type 16 (HPV16) late gene expression. Of the seven SR proteins analyzed here, SRSF1, SRSF3, and SRSF9 induced HPV16 late gene expression, and five of the SR proteins affected HPV16 L1 mRNA splicing. Of the 15 hnRNP proteins analyzed here, hnRNP A2, hnRNP F, and hnRNP H efficiently induced HPV16 late gene expression, and all of the hnRNPs affected HPV16 L1 mRNA levels or mRNA splicing. Thus, the majority of SR proteins and hnRNPs have the potential to regulate HPV16 L1 mRNA splicing. Strict control of the expression of the immunogenic L1 and L2 capsid proteins may contribute to the ability of HPV16 to cause persistence.

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' untranslated regions (3' UTRs) of ORF10-targeted transcripts conferring sensitivity to export inhibition. The Rae1-ORF10 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.

Lepidopteran cells rely on multiple antiviral responses to defend against baculovirus infections, including apoptosis, global protein synthesis shutdown, and rRNA degradation. Here, we characterized apoptosis and rRNA degradation in Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Bombyx mori cells, a system resulting in abortive infection, in relation to viral DNA replication and viral late gene expression. RNAi-mediated silencing of viral DNA replication-related genes prevented apoptosis, but not rRNA degradation, in B. mori cells infected with p35-deficient AcMNPV. Additionally, AcMNPV, but not B. mori nucleopolyhedrovirus (BmNPV), drastically reduced B. mori cellular iap1 transcript levels and p35-deficient AcMNPV induced more prominent apoptosis than did p35-deficient BmNPV. These results, together with previous results that global protein synthesis shutdown follows viral DNA replication, demonstrate that rRNA degradation is the primary antiviral response that abolishes productive AcMNPV infection of B. mori cells. Our results also demonstrate that B. mori cells induce apoptosis to a different extent depending on NPV species.

The model for chickenpox is based on the schema for the pathogenesis of mousepox, which was initially described by the Australian virologist F. Fenner. The number of deaths in chickenpox of infancy was examined in greater detail for the 15 year period of 1968 to 1982. This chapter discusses Neonatal chickenpox because its various manifestations can be correlated with a dual viremic pathogenetic schema. It describes properties of the genomic Varicella Zoster Virus (VZV). One of the most serious manifestations of the extracutaneous replication of virus is varicella pneumonitis. A major structural protein of similar molecular weight has been found in nearly all herpesviruses studied to date. At least 3 prominent glycosylated proteins were also detected in the purified VZV virion preparation. The chapter reviews these structural glycoproteins. Because the VZV late gene products have been most extensively characterized to date, this review emphasizes structural VZV glycoproteins and proteins.

Baculovirus infection follows a transcriptionally controlled sequence of gene expression that occurs by activation of different viral gene promoter sequences during infection. This sequence of promoter activation may be disrupted by cellular defenses against viral infection, which might interfere with viral progeny formation. In this work, the activity of the ie1, gp64, lef-1, vp39, p6.9 and polh promoters of the Anticarsia gemmatalis multiple nucleopolyhedrovirus was assessed during infection of permissive, semipermissive and nonpermissive cell lines by a novel methodology that detects reporter protein luminescence in real-time. This technique allowed us to characterize in rich detail the AgMNPV promoters in permissive cell lines and revealed differential profiles of expression in cells with limited permissivity that correlate well with limitations in viral DNA replication. Semipermissive and nonpermissive cell lines presented delays and restrictions in late and very late promoter expression. Cells undergoing apoptosis did not inhibit late gene expression; however, viral progeny formation is severely affected. This work demonstrates the application of the real-time luminescence detection methodology and how the promoter expression profile may be used to diagnose cellular permissivity to baculovirus infection.

Five proteins are ejected from the bacteriophage T7 virion at the initiation of infection. The three known proteins of the internal core enter the infected cell; all three must both disaggregate from their structure in the mature virion and also almost completely unfold in order to leave the head and pass through the head–tail connector. Two small proteins, the products of genes 6.7 and 7.3, also are ejected from the infecting virion. Gp6.7 and gp7.3 were not previously described as structural virion components, leading to a re-appraisal of the stoichiometry of virion proteins. Gp6.7 is found in tail-less particles and is defined as a head protein, whereas gp7.3 is localized in the tail. Gene 6.7 may be important in morphogenesis; mutants defective in this late gene yield a reduced burst of progeny. Gene 7.3 is essential for virion assembly but, although normally present, its product gp7.3 is not required in a mature particle. Particles assembled in the absence of gp7.3 contain tail fibers but fail to adsorb to cells.

The late expression factor-8 gene (lef-8) of Autographa californica M nucleopolyhedrovirus encodes the largest subunit of the virally encoded DNA-directed RNA polymerase specific for the transcription of late and very late viral genes. The sequence of lef-8 predicts a C-terminal motif of 13 amino acids that is conserved in other polymerases. Detailed mutagenesis throughout lef-8 was performed, including this C-terminal motif, to define sequences required for late promoter activation. It was found that the conserved C-terminal motif was critical for late gene expression. In addition, regions throughout the entire lef-8-encoding sequence were important for optimal function, suggesting complex protein-protein and protein-DNA interrelationships in the late gene-specific viral transcriptosome.

Human papillomaviruses (HPVs) modulate expression of host microRNAs. Our deep-sequencing analysis of organotypic raft cultures identified microRNA 145 (miR-145) as a differentiation-dependent microRNA that has functionally active target sequences in the HPV-31 E1 and E2 open reading frames. Overexpression of miR-145 in HPV-positive cells resulted in reduced genome amplification and late gene expression, along with decreased levels of cellular transcription factor KLF-4. Our studies show that HPV modulates miR-145 expression to control its own life cycle.

This chapter describes a protocol that allows accurate in vitro transcription of vaccinia virus late genes. In this method, extracts are made from vaccinia virus-infected cells and used as enzyme sources to produce mRNAs from plasmid templates containing late gene promoter sequences.

Human trophoblast cells were permissively infected by human cytomegalovirus. The kinetics of viral immediate-early, early, and late gene expression was clearly delayed compared to that in fibroblasts. Productive infection was unequivocally proven by the detection of virion particles, infectious virus in trophoblast culture supernatant, and cell-to-cell spread of cytomegalovirus from infected trophoblasts to uninfected fibroblasts. These observations indicate that infected trophoblasts may be involved in maternofetal transmission of human cytomegalovirus.

Baculovirus replication initiates a cascade of gene expression that ultimately results in the production of progeny virus. This cascade is regulated at different points during the replication cycle: early gene expression is regulated by the interaction of cis-acting elements, viral trans-acting factors, and host factors (see Chapter 6, this volume), and late/very late gene expression is regulated by a combination of viral DNA replication, cis-acting elements as well as early and late viral factors (see Chapter 8, this volume).

The late stage of baculovirus infection is defined as those events that occur following the initiation of viral DNA replication and is usually subdivided into late and very late phases. These phases coincide with the production of the budded and occluded forms of the virus, respectively (see Chapters 2 and 4, this volume). Budded viruses (BVs) are produced by the packaging of newly synthesized viral DNA into nucleocapsids that leave the nucleus and bud through the cell’s plasma membrane. Occluded viruses (OVs) are produced later when the nucleocapsids become enveloped within the nucleus; the resulting virions are then embedded in a crystalline protein matrix, thereby forming nuclear bodies known as polyhedra or granules, depending on their morphological appearance, which, in turn, specifies their genus (nucleopolyhedrosis virus or granulosis virus, respectively).

The transcriptional characterization of the gene coding for the p12 attachment protein of the African swine fever virus is presented. The results obtained have been used to generate the first detailed transcriptional map of an African swine fever virus late gene. Novel experimental evidence indicating the existence of major differences between the mechanisms controlling the transcription of late genes in African swine fever virus and poxviruses is provided.

The drastically reduced virus yields obtained from monkey cells abortively infected with adenovirus 2 (Ad2) have been attributed primarily to a severe decrease in the accumulation of the virion protein fiber (IV), a product of the most pomoter distal late gene family, L5. Here we report that the accumulation of virion protein IIIa, a product of the proximal late gene family, L1, is also severely depressed. In contrast, the i-leader protein LO-13.6K and L1 protein(s) 52K/55K are expressed with the same time course and in equal amounts in monkey cells abortively infected by Ad2 or productively infected by the Ad2-simian virus 40 (SV40) hybrid Ad2+ND1 or by the host range mutant Ad2+ND3 hr602. L1-52K/55K is phosphorylated in abortively infected CV-1 or CV-C monkey cells as well as in productively infected human and monkey cells. As with fiber expression, the failure to produce IIIa appears to be due partly to reduced or delayed IIIa mRNA accumulation. The small amount of IIIa protein that is synthesized in monkey cells is stable. Since the accumulation of both IIIa and fiber protein is deficient, the mechanism of abortive infection cannot be attributed solely to the absence of the auxiliary fiber leader sequences (1).

A decrease in production of bacteriophage T7 was observed in bleomycin-treated Escherichia coli B cells. Bleomycin was found to shorten the eclipse in phage growth. A T7 early gene product, the T7-specific RNA polymerase which catalyzed the transcription of late gene appeared more rapidly in the bleomycin-treated cells than in the non-treated cells. The rate of phage adsorption increased to some extent in drug-treated cells. A possible mechanism to explain the mode of action of bleomycin is discussed.

A total of 103 amber mutants of coliphage P1 were tested for lysis of nonpermissive cells. Of these, 83 caused cell lysis at the normal lysis time and have defects in particle morphogenesis. Five amber mutants, with mutations in the same gene (gene 2), caused premature lysis and may have a defect in a lysis regulator. Fifteen amber mutants were unable to cause cell lysis. Artificially lysed cells infected with five of these mutants produced viable phage particles, and phage particles were seen in thin sections of unlysed, infected cells. However, phage production by these mutants was not continued after the normal lysis time. We conclude that the defect of these five mutants is in a lysis function. The five mutations were found to be in the same gene (designated gene 17). The remaining 10 amber mutants, whose mutations were found to be in the same gene (gene 10), were also unable to cause cell lysis. They differed from those in gene 17 in that no viable phage particles were produced from artificially lysed cells, and no phage particles were seen in thin sections of unlysed, infected cells. We conclude that the gene 10 mutants cannot synthesize late proteins, and it is possible that gene 10 may code for a regulator of late gene expression for P1.

Deletion mutants of bacteriphage T5 can be identified by analyzing the DNA content of single plaques, after alkaline denaturation, by agarose gel electrophoresis. Use of this procedure has resulted in the isolation of a new mutant, designated del2, that lacks a 1.6% segment located 68 to 70% from the left end of T5 DNA. This segment occurs between the early and late gene regions of T5 DNA.

Growth of lymphoblastoid cells (B95-8, Raji) is not inhibited by the presence of 0.4 mM phosphonoacetic acid. The synthesis of Epstein-Barr virus (EBV) in the producer line B95-8 is completely inhibited, as shown by the total inhibition of viral capsid antigen synthesis. Early viral antigens are made normally in the presence of phosphonoacetic acid, but EBV DNA synthesis is blocked in cells entering the productive cycle. Nonproducer cells in the population replicate the resident EBV DNA by a mechanism that is resistant to phosphonoacetic acid. These results are consistant with the hypotheses that EBV DNA is replicated by two mechanisms, one in the noninduced cell and a different mechanism in the producer cell, and that prior replication of EBV DNA, probably by the second mode, is a prerequisite for late gene expression.

A λ mutant has been isolated which is able to grow in the absence of the product of the N gene. The properties of this mutant suggest that the only role of the N gene is to promote the functioning of early λ genes, including gene Q, and that the Q product is then sufficient for synthesis of late gene products.

Infection of fibroblasts by human cytomegalovirus (HCMV) rapidly activates the NF-kappaB signaling pathway, which we documented promotes efficient transactivation of the major immediate-early promoter (DeMeritt, I.B., Milford, L.E., Yurochko, A.D. (2004). Activation of the NF-kappaB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78, 4498-4507). Because a second, sustained increase in NF-kappaB activity following the initial phase of NF-kappaB activation was also observed, we investigated the role that this prolonged NF-kappaB activation played in viral replication and late gene expression. We first investigated HCMV replication in cells in which NF-kappaB activation was blocked by pretreatment with NF-kappaB inhibitors: HCMV replication was significantly decreased in these cultures. A decrease in replication was also observed when NF-kappaB was inhibited up to 48 h post-infection, suggesting a previously unidentified role for NF-kappaB in the regulation of the later class of viral genes.

The late genes of the temperate Salmonella phage P22 comprise an operon. The late operon of P22 contains within it a gene ( 9 ) which is separated from the other late genes by a set of genes ( imm I) which are regulated independently of the late genes. These results were obtained by exploiting the orientation-dependent polarity of insertions of the translocatable ampicillin-resistance element Tn1. The data are consistent with a model of P22 late gene regulation in which the positive activator of the late operon, gene 23 , can act as a selective antagonist of transcription termination.

We have examined the organization and properties of the genes around the region controlling late gene expression in phage 82. We have isolated mutants of λ(QSR)82 hybrid phages that are analogous to λ Q−, S−, and R− mutants; they map on the 82 genome in the same order as the corresponding λ mutations. The QSR region of 82 encodes a small, r-strand-specific transcript which is probably the 82 analog of the λ 6 S RNA, and thus may be involved in the regulation of late gene expression in 82.